Remedy for ischemia

ABSTRACT

The present invention provides a ischemia therapeutic agent that contains vascularization induction factor and gelatin hydrogel, and gradually releases vascularization induction factor, which is useful in treating ishchemia accompanying peripheral circulatory disorders etc. encountering as complications of arteriosclerosis obliterans, Buerger&#39;s disease, diabetes and collagen disease.

TECHNICAL FIELD

The present invention relates to a ischemia therapeutic agent thatcontains a vascularization induction factor and a gelatin hydrogel, andgradually releases vascularization induction factor.

BACKGROUND ART

In the field of vascular surgery, numerous cases have been reportedinvolving serious ischemia of the upper or lower extremities caused byperipheral vasculitis associated with atherosclerosis obliterans,thromboangitis obliterans (Buerger's disease) and collagen disease.

Although serious upper or lower extremity ischemic diseases as such arenot lethal, the resulting amputation of an upper or lower extremitycauses immense psychological and physical suffering for the patient.

Moreover, serious ischemia of the extremities not only causes poorcirculation, but also causes delayed wound healing and refractoryinfections at the affected area, and for example, can cause infection ofartificial blood vessels in patients who have undergone bypass surgeryusing artificial blood vessels, which can ultimately lead to a fataloutcome.

However, there are currently no known treatment methods for thiscondition that have demonstrated adequate therapeutic effects.

A known example of a surgical treatment method is lower extremityvascular reconstruction. In addition, the application range of thistreatment method is tending to expand to also include elderly patientsand patients complicated with diseases involving other organs, for whichit was difficult to apply this treatment in the past. However, a largenumber of unsuccessful cases of have been reported due to increases inthe patient population resulting from improved diagnostic techniques,and there have even been cases reported which have ultimately resultedto upper or lower extremity amputation. Namely, in serious cases, thetherapeutic effects of surgical treatment methods are only able tosomewhat extend the period during which the extremities are able to beconserved, and their treatment results have been extremely poor.

On the other hand, although medicinal treatment methods primarilyconsist of promotion of collateral circulation through administration ofcirculation ameliorants such as vasodilators, there is currently noknown treatment method for which adequate effects can be obtained.

Treatment methods for ischemic diseases are recently being developedthat utilize vascularization induction factors.

Vascularization induction factors (vascularization promotion factors)are observed in mature individuals during the course of the progressionof a disease state such as in wound healing, growth and metastasis ofsolid cancers, chronic inflammations and retinopathy. These factorspromote the destruction of the basal membranes of venules followingexisting capillaries, germination of endothelial cells from areas oflocal destruction, migration and proliferation outside vessels, andvarious processes of lumen formation, and have activity that promotesthe formation of new capillaries and small blood vessels. Typicalexamples of these factors include basic fibroblast growth factor (bFGF),vascular endothelial growth factor (VEGF), hepatocyte growth factor(HGF), angiotensin, platelet-derived growth factor (PDGF) and ephrin.bFGF is used in the US and Europe a the clinical treatment level for thetreatment of ischemic heart disease. In addition, bFGF is also used inJapan at the clinical application level in the field of dermatology, andat the clinical trial level in the field of orthopedic surgery.

A treatment method for ischemic diseases has also been developed thatuses genes such as VEGF and HGF. This treatment method consists ofadministering the gene primarily into muscle, causing the gene to beincorporated in muscle cells, and causing the expression product of theinserted gene in the form of protein to be secreted from the cellscontaining the gene. This method is characterized by gradual releaseusing cells, or in other words, causing cells to gradually release avascularization induction factor. However, this method has theshortcomings of the gene expression efficiency being low, and beingunable to control the level or timing of gene expression. In addition,there is also the problem of the expression of unknown effects resultingfrom gene insertion still not having been resolved.

In other words, the most important factor in terms of solving theaforementioned problems is the gradual release of vascularizationinduction factor. The reason for attempting to secrete a cell growthfactor from cells using a gene and obtain the effects of its gradualrelease is that, in the case of administering a vascularizationinduction factor in the form of an aqueous solution, expression of theaction of the vascularization induction factor is not observed at all,and the vascularization induction factor itself is unable to begradually released.

If it were possible to gradually release a cell growth factor as in thepresent invention however, it would be meaningless to select a methodthat uses a gene, and the aforementioned problems would be able to besolved.

The inventors of the present invention surprisingly found that apreparation that contains a vascularization induction factor and agelatin hydrogel and gradually releases vascularization induction factoris useful for the treatment of ischemia of the upper or lowerextremities, and that a treatment method using this preparation is lessinvasive and more potently increases blood flow to seriously affectedupper and lower extremities by promoting collateral circulation ascompared with known treatment methods of the prior art, thereby leadingto completion of the present invention.

DISCLOSURE OF THE INVENTION

Thus, the object of the present invention is to provide an ischemiatherapeutic agent that contains vascularization induction factor andgelatin hydrogel, and gradually releases vascularization inductionfactor.

A gelatin used in the present invention differs from commerciallyavailable gelatin and is a gelatin that has the following physicalproperties:

(1) an acidic gelatin obtained from collagen by alkaline hydrolysistreatment;

(2) molecular weight under non-reducing conditions of SDS-PAGE of about100,000 to about 200,000 daltons; and,

(3) the zeta potential in aqueous solution of about −15 to about −20 mV.

Although examples of commercially available gelatins include the type Agelatin manufactured by Sigma and the gelatin manufactured by Wako PureChemical Industries, the zeta potential in aqueous solution differs inthe manner shown below.

Sigma Type A Gelatin: Roughly 0 to roughly 5 mV

Wako Gelatin: Roughly −5 to roughly −2 mV

The zeta potential is an indicator that represents the degree ofelectrostatic charge of a substance (gelatin), and is considered to besuitable as an indicator of a gelatin that forms an electrostaticcomplex with HGF in the present invention.

A gelatin of the present invention is obtained by alkaline hydrolysisfrom a part such as the skin or tendon of various animal species such ascows, from collagen, or from a substance used as collagen. Preferably,it is an acidic gelatin prepared by alkaline treatment of type Icollagen originating in bovine bone, and can also be acquired having asample isoelectric point (IEP) of 5.0 from Nitta Gelatin. Furthermore,although basic gelatin prepared by acid treatment can also be similarlyacquired from Nitta Gelatin having an IEP of 9.0, the zeta potential isconsiderably different as indicated below.

-   -   Acidic gelatin (Nitta Gelatin sample IEP 5.0): Roughly −15 to        roughly −20 mV    -   Basic gelatin (Nitta Gelatin sample IEP 9.0): Roughly +12 to        roughly +15 mV

A gelatin hydrogel used in the present invention refers to a hydrogelobtained by using the aforementioned gelatin and condensing with variouschemical crosslinking agents. Examples of chemical crosslinking agentsthat can be used include glutaraldehyde, EDC and other water-solublecarbodiimides, propylene oxide, diepoxy compounds and condensationagents. An example of a chemical crosslinking agent that is usedpreferably is glutaraldehyde.

In addition, the gelatin can also be crosslinked by heat treatment orultraviolet irradiation.

There are no particular limitations on the form of the gelatin hydrogel,and examples include cylinders, square columns, sheets, disks, spheresand particles. Gelatin hydrogels in the form of cylinders, squarecolumns, sheets and disks are frequently used as implants, while spheresand particles can also be administered by injection.

Gelatin hydrogels in the form of cylinders, square columns, sheets anddisks can be prepared by adding a crosslinking agent aqueous solution toa gelatin aqueous solution or adding gelatin to a crosslinking agentaqueous solution, followed by pouring into a mold of a desired shape andallowing the crosslinking reaction to proceed. In addition, a moldedgelatin gel may be added directly or after drying to a crosslinkingagent aqueous solution. The crosslinking reaction is stopped bycontacting with a low molecular weight substance having an amino groupsuch as ethanol amine or glycine, or by adding an aqueous solutionhaving a pH of 2.5 or lower. The resulting gelatin hydrogel is used toprepare a preparation after washing with distilled water, ethanol,2-propanol or acetone and so forth.

A gelatin hydrogel in the form of spheres or particles can be preparedby, for example, attaching an immobilized stirring motor (for example,the 3-1 motor, EYELA mini D.C. stirrer, manufactured by ShintoScientific) and Teflon (trade name) propeller to a three-mouth,round-bottom flask, placing the flask and immobilized apparatus in agelatin solution, adding an oil such as olive oil, stirring at a speedof about 200 to 600 rpm to form a W/O emulsion and adding a crosslinkingagent aqueous solution thereto, or dropping the product ofpre-emulsifying a gelatin aqueous solution in olive oil (for example,using the Advantec 21 vortex mixer, homogenizer or polytron PT10-35)into olive oil to prepare a fine particulate W/O emulsion, followed bythe addition of a crosslinking agent aqueous solution and allowing thecrosslinking reaction to proceed. After then recovering the gelatinhydrogel by centrifugal separation, it is washed with ethyl acetate andso forth followed by immersing in 2-propanol or ethanol to stop thecrosslinking reaction. The resulting gelatin hydrogen particles are usedto prepare a preparation after sequentially washing with 2-propanol,Tween 80-containing distilled water and distilled water.

In the case the gelatin hydrogel particles aggregate, the addition of,for example, a surfactant or ultrasonic treatment (preferably for nomore than about 1 minute while cooling) may be carried out.

Furthermore, a fine particulate gelatin hydrogel having a particle sizeof 20 μm or less can be obtained by pre-emulsification.

The mean particle size of the resulting gelatin hydrogel particles is 1to 1000 μm, and these particles should be used after sizing to therequired size according to the purpose of use.

The following provides an example of another method of preparing agelatin hydrogel in the form of spheres or particles.

After placing olive oil in the same apparatus as used in the previousexample, stirring at a speed of about 200 to 600 rpm, dropping in agelatin aqueous solution to prepare a W/O emulsion and cooling, ethylacetate and so forth is added and stirred followed by recovering thegelatin particles by centrifugal separation. After additionally washingthe recovered gelatin particles with acetone and ethyl acetate, and thenwith 2-propanol and ethanol, etc., the particles are allowed to dry. Thedry gelatin particles are then suspended in a crosslinking agent aqueoussolution containing 0.1% Tween 80, the crosslinking reaction is allowedto proceed while stirring gently, and the particles are washed with 100mM glycine aqueous solution containing 0.1% Tween 80 or 0.004 N HClcontaining 0.1% Tween 80 depending on the crosslinking agent usedfollowed by stopping the crosslinking reaction to prepare gelatinhydrogel particles. The mean particle size of the gelatin hydrogelparticles obtained with this method is similar to that in the case ofthe aforementioned method.

The mechanism of this gradual release is based on vascularizationinduction factor being physically immobilized on gelatin within thehydrogel. In this state, the factor is not released from the hydrogel.If the gelatin molecules become soluble in water as a result of thehydrogel being decomposed, the immobilized vascularization inductionfactor is released accompanying that decomposition. Namely, the gradualrelease properties of the vascularization induction factor can becontrolled by the decomposition of the hydrogel. The ease ofdecomposition of the hydrogel can be changed according to the degree ofcrosslinking during preparation of the hydrogel.

There are no particular limitations on the conditions of thecrosslinking reaction, and it can be carried out, for example, at 0 to40° C. for 1 to 48 hours.

A gelatin hydrogel of the present invention is such that its moisturecontent clearly has a considerable effect on the gradual releaseproperties of the vascularization induction factor, and an example of amoisture content that demonstrates preferable gradual release effects isabout 80 to 99 w/w %, while a more preferable moisture content is about95 to 98 w/w %. Moisture content can be used as a measurable indicatorof the degree of crosslinking. A large moisture content indicates a lowdegree of crosslinking as well as greater susceptibility todecomposition. In other words, the value of this moisture contentaffects the gradual release properties of the vascularization inductionfactor.

A gelatin hydrogel of the present invention can be used after suitablycutting to an appropriate size and shape, freeze-drying and sterilizing.Freeze-drying can be carried out by, for example, placing the gelatinhydrogel in distilled water, freezing for 30 minutes or more in liquidnitrogen or for 1 hour or more at −80° C., and then drying for 1 to 3days in a freeze-dryer.

Although the concentrations of gelatin and crosslinking agent whenpreparing a gelatin hydrogel should be suitably selected according tothe desired moisture content, an example of the gelatin concentration is1 to 20 w/w %, while an example of the crosslinking agent concentrationis 0.01 to 1 w/w %.

The vascularization induction factor used in the present invention is aknown substance, and that prepared by various methods can be usedprovided it has been purified to a degree that allows it to be used as abiochemical reagent or pharmaceutical. In addition, a commerciallyavailable product (such as Fiblast Spray®) may also be used. An exampleof a method for producing vascularization induction factor consists ofculturing primary cultured cells or established cells that producevascularization induction factor, separating from the culturesupernatant and so forth, and purifying to obtain vascularizationinduction factor. Alternatively, a gene that encodes vascularizationinduction factor can be incorporated in a suitable vector using geneticengineering techniques followed by transformation of a suitable host byinserting in said host, and then obtaining the target recombinantinduction factor from the culture supernatant of the transformants.There are no particular limitations on the aforementioned host cells,and various host cells conventionally used in genetic engineeringtechniques can be used, examples of which include E. coli, yeast andanimal cells. The induction factor obtained in this manner may have oneor multiple amino acids in its amino acid sequence substituted, deletedand/or added, or a sugar chain may be similarly substituted, deletedand/or added, provided it has substantially the same action asnaturally-occurring induction factor.

Although any vascularization induction factor can be used for thevascularization induction factor used in the present invention providedit has activity that promotes formation of new capillaries, examples ofsuch vascularization induction factors include basic fibroblast growthfactor (bFGF), vascular endothelial growth factor (VEGF), hepatocytegrowth factor (HGF), angiotensin, platelet-derived growth factor (PDGF)and ephrin.

A vascularization induction factor gradual release gelatin hydrogelpreparation in the present invention refers to a preparation that isobtained by immersing a vascularization induction factor into theaforementioned acidic gelatin hydrogel. Although vascularizationinduction factor forms a complex with acidic gelatin hydrogel because itis a basic protein, when considering the absorption inhibitory effectsof bFGF with respect to the aforementioned changes in ionic strength insolution, this vascularization induction factor gelatin (hydrogel)complex not only involves electrostatic interaction, but is alsoaffected by other interactions such as hydrophobic bonding. Thedissociation constant (Kd) of this complex as well as the binding molarratio of the induction factor to gelatin were obtained according to aScatchard binding model (Scatchard, G., 1949). The binding molar ratioof bFGF to gelatin is such that roughly 1 bFGF molecule binds to 1acidic gelatin molecule.

In addition, the Kd value of acidic gelatin at 37° C. is 5.5×10⁻⁷ M,which is about two to three orders larger than the Kd value of heparinsulfate at 20° C. of 1×10⁻⁹ to 2.0×10⁻¹⁰ M (Rahmoune, H. et al., 1988).This indicates that binding of the HGF gelatin complex is weak and notas strong as that between HGF and heparin sulfate.

In the case the molar ratio of a vascularization induction factor suchas bFGF, for example, to gelatin is about 1:1 or more, liberation ofbFGF occurs easily and the resulting behavior is quite similar to thatof free bFGF in terms of activity. However, in the case the molar ratioof bFGF to gelatin is lowered to about 1:1 or less, since the bFGF isadsorbed and the amount that is liberated is reduced, the apparentactivity of bFGF appears to decrease.

Thus, although a complex of vascularization induction factor and gelatinor gelatin hydrogel can be made in which the molar ratio between thevascularization induction factor and gelatin is changed in various ways,in order to avoid an initial burst, a preferable example of a complexhas a molar ratio in which there are about 1 mole or less ofvascularization induction factor to 1 mole of gelatin hydrogel.

Furthermore, the weight ratio of vascularization induction factor togelatin is preferably about 5 or less, and the weight ratio ofvascularization induction factor to gelatin is more preferably about 5to about 1/10⁴.

Since a vascularization induction factor gradual release gelatinhydrogel preparation of the present invention has vascularizationinduction factor gradual release effects and stabilizing effects, it isable to demonstrate the function of a vascularization induction factorfor a long period of time even in small amounts. Consequently, theinherent function of vascularization induction factors in the form ofcardiovascular protective action such as promotion of vascularization,prevention of reperfusion injury and inhibition of fibrosis are able tobe demonstrated effectively.

A vascularization induction factor gelatin hydrogel preparation of thepresent invention can be used parenterally as an injection preparation.It can be administered, for example, subcutaneously, intramuscularly,intravenously, intracelomicly, into connective tissue, intraperiosteallyor into a damaged organ.

A vascularization induction factor gradual release gelatin hydrogelpreparation of the present invention or complex thereof can be used in asuitable drug form according to the respective application. For example,it can be administered in a drug form such as a sheet, stick, particles,rods or paste. Examples of administration methods includeintracutaneous, subcutaneous, intramuscular, intracelomic, intoconnective tissue and intraperiosteal administration.

Although the dosage of vascularization induction factor in a preparationof the present invention can be suitably adjusted according to thepatient's severity, patient's age and body weight, etc., the normaladult dosage is selected from the range of about 0.1 to about 500 μg,and preferably from the range of about 1 to about 100 μg, and can beinjected into the affected area or a peripheral site thereof. Inaddition, said administration can be performed a plurality of times inthe case effects are inadequate with a single administration.

A preparation according to the present invention can be applied totreatment of ischemia in the field of vascular surgery. This ischemia ispreferably ischemia accompanying a disease selected from the groupconsisting of atherosclerosis obliterans, Buerger's disease and poorperipheral circulation occurring as a complication of diabetes orcollagen disease.

Although a preparation according to the present invention can be appliedto the treatment of ischemia caused by poor peripheral circulation, itis preferably applied to the treatment of ischemia in the upper or lowerextremities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the normal course of development of collateral circulationin an untreated group designated as group A. Although there aredifferences between individuals, development of collateral circulationis observed that is the result of intrinsic growth factors even in theabsence of treatment. Pre indicates prior to treatment, while postindicates four weeks after treatment.

FIG. 2 shows the development of collateral circulation in a groupadministered gelatin hydrogel only designated as group B. Thedevelopment of collateral circulation is observed to the same extent asthat observed in group A. Pre indicates prior to treatment, while postindicates four weeks after treatment.

FIG. 3 shows the development of collateral circulation in a groupadministered gelatin hydrogel containing bFGF (30 μg) designated asgroup C. The prominent development of collateral circulation paths areobserved as a result of administration of bFGF. Pre indicates prior totreatment, while post indicates four weeks after treatment.

FIG. 4 shows the development of collateral circulation in a groupadministered gelatin hydrogel containing bFGF (100 μg) designated asgroup D. Prominent collateral circulation and increased vascularizationare observed as a result of administration of bFGF. Pre indicates priorto treatment, while post indicates four weeks after treatment.

FIG. 5 shows tissue specimens of muscle sampled from the femoral region(hematoxylin-eosin staining, magnification factor: 20×). In a comparisonbetween group A and group D, in contrast to extensive capillaryformation being observed in group D, capillary formation in group A isobserved to be deficient.

FIG. 6 shows the capillary densities of each group (number ofcapillaries per unit surface area). Significant increases in capillarydensity are observed that are dependent on the dosage of bFGF (levels ofsignificance: group A vs. D=p<0.0001, group B vs. group D=p<0.0001,group C vs. group D=p<0.0001, group A vs. group C=p<0.05)

EXAMPLES

A. Preparation of bFGF-Containing Gelatin Hydrogel

Gelatin hydrogel containing bFGF was prepared according to the methoddescribed in WO 94/27630. More specifically, a mixture ofalkaline-treated gelatin aqueous solution having an isoelectric point of4.9 (10 wt %, 20 ml) and olive oil (5 ml) was preheated at 40° C. andstirred for 1 hour, and after removing the natural gelatin by coolingthe prepared emulsion with ice, acetone was added followed by stirringfor 1 hour at 4° C. The resulting gelatin particles were washed threetimes with acetone (4° C.) and then recovered by centrifugal separation(5000 rpm, 4° C., 5 minutes).

The resulting non-crosslinked gelatin particles (20 mg) were suspendedin an aqueous solution of Tween 80 (0.1%, 20 ml) containingglutaraldehyde (0.13 wt %) and then stirred for 24 hours at 4° C. tocarry out the crosslinking reaction. After recovering by centrifugalseparation (5000 rpm, 4° C., 5 minutes), the particles were stored for 1hour at 37° C. in glycine aqueous solution (20 ml, 10 mM) and washedthree times with distilled water followed by freeze-drying. The meanparticle size of the resulting crosslinked gelatin particles was 10 μm.In addition, the moisture content was 95 w/w %.

Human bFGF described in FIG. 4 of WO 87/01728 was used for the bFGF, andimmersed in the crosslinked gelatin particles by dropping a bFGF aqueoussolution (5 mg, 20 μl) into 2 mg of the freeze-dried gelatin particlesand allowing to stand for 1 hour at room temperature.

B. Study Using Atherosclerosis Obliterans (ASO) Rabbit Model

1) Preparation of ASO Rabbit Model

The common femoral artery of the right hind leg was ligated on theinside under venous anesthesia and local anesthesia using Japanese whiterabbits having body weights of 2.5 to 3.5 kg (males, purchased fromShimizu Laboratory Animals) and then ablated for about 2 cm towards thedistal side. The common femoral artery was then also ligated on thedistal side and the intermediate portion was completely excised toprepare an ASO model.

Since human ASO is a chronic disease, a two-week progress observationperiod was established to create a condition that corresponded to thisdisease.

2) Treatment Method Using bFGF-Containing Gelatin Hydrogel

Angiography was performed on the affected hind legs of all animals inthe second week after surgery and used for an evaluation control forcomparing the status of lower limb circulation following treatment. Theanimals were divided into the following four groups followingangiography, and a different treatment was performed on each group.

Group A (n=6): Not treated (control group)

Group B (n=6): Administration of gelatin hydrogel only

Group C (n=6): Administration of gelatin hydrogel containing bFGF (30μg)

Group D (n=6): Administration of gelatin hydrogel containing bFGF (100μg)

Administration conditions consisted of administration by intramuscularinjection into the femoral region of the affected hind leg for thepurpose of local exposure to bFGF by gradual exposure for four weeks.

3) Evaluation

Angiography was performed on the affected hind leg following a four-weektreatment period, and a sample of muscle was taken from the femoralregion of the affected hind leg for histological evaluation.

i) Angiographic Evaluation

The development of collateral circulation was evaluated for each group.

In group A serving as the untreated group, although there weredifferences between individual animals, development of collateralcirculation due to intrinsic growth factors was observed even thoughthese animals were not treated. In group B that was administered onlygelatin hydrogel, collateral circulation was observed to have developedto about the same degree as that observed in group A. In group C thatwas administered gelatin hydrogen gel containing bFGF (30 μg), prominentdevelopment of collateral circulation paths was observed as a result ofadministration of bFGF. In group D that was administered gelatinhydrogel containing bFGF (100 μg), prominent development of collateralcirculation as well as increased vascularization were observed as aresult of administration of bFGF.

ii) Histological Evaluation

Tissue specimens of muscle sampled from the femoral region(hematoxylin-eosin staining, magnification factor: 20×) are shown inFIG. 5. In contrast to extensive capillary formation being observed ingroup D, capillary formation in group A was observed to be deficient.Capillary densities between each group (number of capillaries per unitsurface area) increased significantly dependent on the dosage of bFGF(FIG. 6).

1. An ischemia therapeutic agent that contains vascularization inductionfactor and gelatin hydrogen, and gradually releases vascularizationgrowth factor.
 2. An ischemia therapeutic agent according to claim 1,wherein the gelatin has the following physical properties: (1) an acidicgelatin obtained from collagen by alkaline hydrolysis treatment; (2)molecular weight under non-reducing conditions of SDS-PAGE of about100,000 to about 200,000 daltons; and, (3) zeta potential in aqueoussolution of about −15 to about −20 mV.
 3. An ischemia therapeutic agentaccording to claim 1, wherein the vascularization induction factor isselected from the group consisting of basic fibroblast growth factor,vascular endothelial growth factor and hepatocyte growth factor.
 4. Anischemia therapeutic agent according to claim 1, wherein the ischemiaaccompanies a disease selected from the group consisting ofatherosclerosis obliterans, Buerger's disease and poor peripheralcirculation occurring as a complication of diabetes or collagen disease.5. An ischemia therapeutic agent according to any of claim 1, whereinthe ischemia is ischemia in the upper or lower extremities.
 6. Anischemia therapeutic agent according to claim 2, wherein thevascularization induction factor is selected from the group consistingof basic fibroblast growth factor, vascular endothelial growth factorand hepatocyte growth factor.
 7. An ischemia therapeutic agent accordingto claim 2, wherein the ischemia accompanies a disease selected from thegroup consisting of atherosclerosis obliterans, Buerger's disease andpoor peripheral circulation occurring as a complication of diabetes orcollagen disease.
 8. An ischemia therapeutic agent according to claim 3,wherein the ischemia accompanies a disease selected from the groupconsisting of atherosclerosis obliterans, Buerger's disease and poorperipheral circulation occurring as a complication of diabetes orcollagen disease.
 9. An ischemia therapeutic agent according to claim 6,wherein the ischemia accompanies a disease selected from the groupconsisting of atherosclerosis obliterans, Buerger's disease and poorperipheral circulation occurring as a complication of diabetes orcollagen disease.
 10. An ischemia therapeutic agent according to claim2, wherein the ischemia is ischemia in the upper or lower extremities.11. An ischemia therapeutic agent according to claim 3, wherein theischemia is ischemia in the upper or lower extremities.
 12. An ischemiatherapeutic agent according to claim 6, wherein the ischemia is ischemiain the upper or lower extremities.
 13. An ischemia therapeutic agentaccording to claim 4, wherein the ischemia is ischemia in the upper orlower extremities.
 14. An ischemia therapeutic agent according to claim7, wherein the ischemia is ischemia in the upper or lower extremities.15. An ischemia therapeutic agent according to claim 8, wherein theischemia is ischemia in the upper or lower extremities.
 16. An ischemiatherapeutic agent according to claim 9, wherein the ischemia is ischemiain the upper or lower extremities.